The USS Klakring (FFG 42) frigate entered the Black Sea on March 20 and is closely monitored by Russia, whose Black Sea Fleet has its main base in Sevastopol. The U.S. warship is scheduled to stay in the city until Saturday before heading to Batumi in Georgia.
"We welcome all foreigners who come here as tourists, but we will not tolerate the U.S. military in our city," said city council member Andrei Merkulov, one of the organizers of the rally.
Locals in Ukraine's Crimea, which has a largely Russian-speaking population, frequently protest against visits by NATO ships.
Last November, the USS Mount Whitney cut short its visit to Sevastopol amid similar protests.
Ukraine has been pursuing NATO membership since pro-Western President Viktor Yushchenko was inaugurated in January 2005. However, regular opinion polls show that the majority of Ukrainians continue to oppose NATO membership.